Mote Marine Laboratory Red Tide Studies

MOTE MARINE LABORATORY RED TIDE STUDIES

FINAL REPORT FL DEP Contract MR 042

July 11, 1994 - June 30, 1995

Submitted To: Dr. Karen Steidinger Florida Marine Research Institute FL DEPARTMENT OF ENVIRONMENTAL PROTECTION 100 Eighth Street South East St. Petersburg, FL 33701-3093

Submitted By: Dr. Richard H. Pierce Director of Research MOTE MARINE LABORATORY 1600 Thompson Parkway Sarasota, FL 34236

Mote Marine Laboratory Technical Report No. 429 June 20, 1995

This document is printed on recycled paper

Suggested reference Pierce RH. 1995. Mote Marine Red Tide Studies July 11, 1994 - June 30, 1995. Florida Department of Environmental Pro- tection. Contract no MR 042. Mote Marine Lab- oratory Technical Report no 429. 64 p. Available from: Mote Marine Laboratory Library. TABLE OF CONTENTS

I. SUMMARY...... 1

II. CULTURE MAINTENANCE AND GROWTH STUDIES ...... 1

Ill. ECOLOGICAL INTERACTION STUDIES ...... 2 A. Brevetoxin Ingestion in Black Seabass B. Evaluation of Food Carriers C. First Long Term (14 Day) Clam Exposure With Depuration (2/6/95) D. Second Long Term (14 Day) Clam Exposure (3/21/95)

IV. RED TIDE FIELD STUDIES ...... 24 A. 1994 Red Tide Bloom (9/16/94 - 1/4/95) B. Red Tide Bloom (4/13/94 - 6/16/95) C. Red Tide Pigment D. Bacteriological Studies E. Brevetoxin Analysis in Marine Organisms Exposed to Sublethal Levels of the 1994 Natural Red Tide Bloom

V. REFERENCES ...... 61

Tables

Table 1. Monthly Combined Production and Use of Laboratory C. breve Culture...... 2

Table 2. Brevetoxin Concentration in Brevetoxin Spiked Shrimp and in Black Seabass Muscle Tissue and Digestive Tract Following Ingestion of the Shrimp ...... 5

Table 3. 14 Day Clam Exposure With Depuration - Water Quality and C. breve Concentrations ...... 7

Table 4. Clam Exposure - Bioaccumulation: 14 Day Exposure With Depuration Brevetoxin Concentrations in Exposure Water and Clams ...... 11

Table 5. Second 14 Day Clam Exposure - Water Quality and C. breve Concentrations ...... 16

Table 6. Brevetoxin Concentrations in Exposure Media and in Clams Exposed to Live G. breve Cells (Second Clam Exposure) ...... 23

Table 7. Red Tide Field Studies (9/19/94 - 12/30/94) ...... 25

Table 8. Brevetoxin Analysis from Natural Red Tide Bloom Areas ...... 33

Table 9. Red Tide Field Studies (4/13/95 - 6/16/95) ...... 34

Table 10. Bacteriological Samples Collected From Gulf and Bay Waters for the 1994/1995 Red Tide Project ...... 48

Table 11. Genera/Groups of Bacteria Recovered From Plate Counts ...... 50

Table 12. Vibrio spp. Recovered From Direct Plating (TCBS) and MPN Tubes ...... 51

Table 13. Oyster, Seasquirt and Mullet Analysis for 9/94 - 1/95 Red Tide Natural Bloom ... 53

i (Table of Contents Continued)

Figures

Figure 1. G. breve Test Tank Cell Counts at 24 Hours for a 7 Day Clam Exposure ...... 13

Figure 2. G. breve Test Tank Cell Counts at 24 Hours for a 14 Day Clam Exposure ...... 14

Figure 3. G. breve Cell Counts for Individual Exposure - Tanks After 24 Hours ...... 21

Figure 4. T. chuii Cell Counts for individual - Control Tank Counts After 24 Hours ...... 22

Figure 5. New Pass Dock Cell Concentrations, During the 1994 Red Tide Bloom ...... 32

Figure 6. Red Tide Cell Count; New Pass, 1995 Red Tide Bloom ...... 41

Figure 7. Chlorophyll a Content of G. breve at Two Samplings Depths During a Natural Red Tide Bloom. September - December, 1994 ...... 45

Figure 8. Gyroxanthin-diester Content of G. breve at Two Sampling Depths During a Natural Red Tide Bloom. September - May 1994 ...... 46

Figure 9. Comparison of the Concentration of Three Pigments with Chlorophyll, a Concentration in G. breve Collected at 2m Depth During a Natural Red Tide Bloom ...... 47

Figure 10. Red Tide Toxin (BTX) in Oysters & Seasquirts During a Natural Red Tide Bloom in Sarasota Bay ...... 60

Appendixes

Appendix “A” Methods for Obtaining Phytoplankton Cell Counts ...... 62

Participants

Mote Marine Laboratory

Dr. Richard Pierce ...... Project Manager Dr. Gary Kirkpatrick ...... Phytoplankton Ecology/Pigment Analysis Dr. John Buck ...... Microbiology Mr. Mike Henry ...... Toxin Analysis/Field Monitoring Ms. Jing Zhou ...... Toxin Analysis/Field Monitoring Ms. Cindy Aller ...... Culture Maintenance/Bioaccumulation/Field Monitoring Ms. Tammy Seaman ...... Culture Maintenance/Bioaccumulation/Field Monitoring

National Marine Fisheries Service, Charleston Laboratory

Dr. Fran VanDolah ...... Receptor Binding Assay for Brevetoxins Mr. Todd Leighfield ...... Receptor Binding Assay for Brevetoxins

ii 1. SUMMARY

The Mote Marine Laboratory Red Tide Research Program encompasses four study areas:

1. Laboratory culture maintenance and growth; 2. Ecological interactions; 3. Toxin chemistry and analysis; and, 4. Red tide bloom investigations.

These studies, in cooperation with the Florida Department of Environmental Protection, Florida Marine Research institute, focus on the accumulation of red tide toxins in fish and shellfish and the effects of chronic exposure to red tide toxins during natural blooms and under controlled laboratory exposure conditions.

Natural red tide bloom studies included weekly monitoring of the intensity and distribution of the bloom, analysis of toxin and pigment content of intensive bloom areas, marine bacteria associated with the bloom and bioaccumulation of brevetoxins in sea squirts, oysters, clams and mullet collected from an area of Sarasota Bay experiencing chronic, low levels of the red tide bloom.

In addition to natural bloom monitoring, laboratory red tide cultures were maintained for use in exposure - bioaccumulation studies with clams and a fish, black seabass. Tissues were analyzed for toxin content. Extracts of exposed invertebrates were sent to the NOAA-NMFS Southeast Fisheries Science Center in Charleston, SC, for receptor binding analysis.

II. CULTURE MAINTENANCE AND GROWTH STUDIES

Gymnodinium breve culture production was maintained between 120-200 liters throughout the twelve-month period with volume varying according to experimental use. Production and use of G. breve culture are found in Table 1. Cultures were maintained in both artificial (NH-15) media and (f/2) a natural seawater base media (Guillard & Ryther, 1962).

In addition to G. breve, Mote Marine Laboratory maintained other phytoplankton cultures. Presently, Tetraselmis sp., Dunneliella tertiolecta, Thalassiosira psuedonana, T. weissflogii, Iochrysis galbana, Nannochloropsis occulata, and Skeletonema costatum are cultured for pigment comparisons. Tetraselmis chuii is cultured in large volume as a primary food source for clams in bioassay.

Twelve liter polycarbonate carboys were tested as a substitute for fragile glass carboys. The growth and life cycle of G. breve were found to be comparable in both types of carboys. However, in the twenty liter polycarbonate carboys, the stationary phase of G. breve was prolonged. Cultures were maintained without inoculation in these carboys for 279 days. The randomly monitored parameters of dissolved oxygen, pH and salinity demonstrated an extended typical “stationary phase”.

C. breve culture was received from Dr. David Millie, USDA. New Orleans, and was used to simulate repopulation of our culture supply after a facility culture crash. The culture was successfully grown from test tube to carboy in 300 days. Table 1. Monthly combined production and use of laboratory G. breve culture.

Total volume Total volume Net change in of new culture of culture total culture inoculated (L) used (L) volume (L)

Months July 1994 108 174 -66 August 1994 180 102 78 September 1994 54 66 -12 October 1994 180 150 -30 November* 1994 198 186 -12 December 1994 120 120 0 (up to the 21st) January 1995 155.5 151.5 4 February 1995 118 146 -28 March 1995 228 247 -19 ApriI 1995 185 206 -21 May 1995 130 72 +58 June 16th 1995 0 0 0 * in the month of November, the culture room had an AC problem which contributed to loss of culture.

Ill. ECOLOGICAL INTERACTION STUDIES

A. Brevetoxin Ingestion in Black Seabass

1) Materials and Methods

Fish are very susceptible to red tide toxins when they enter the blood stream through the gills. The effect from ingestion, however has not been established. This study was undertaken to investigate the effect of brevetoxins ingested by black seabass and the fate of the ingested toxins.

Black seabass (Centropristis striatus), 16.5 to 20.5 cm in length, were placed three each in (250 L) aquaria and fed a daily diet of commercial fish feed pellets (Ziggler, Inc., P.A.). Brevetoxin was ingested by daily feeding with pieces of shrimp spiked with known amounts of brevetoxins. The brevetoxin concentrations were 0, 100, 250 & 500 µg/day, fed continuously over a 4 day (96 hour) period.

The source of the brevetoxins was cultures of G. breve grown at Mote Marine Laboratory. Brevetoxins were extracted from the cultures and isolated according to the procedure of Pierce et al., 1992, with quantitative analysis performed by HPLC. Known quantities of toxin were injected into pieces of shrimp in methanol solution, and the shrimp were subsequently fed to each fish. Fish were fed about 4 g of shrimp twice daily for four days, with half of the daily toxin dose provided each time. Ingestion of the shrimp was carefully monitored.

2 Table 1. Monthly combined production and use of laboratory G. breve culture.

Total volume Total volume Net change in of new culture of culture total culture inoculated (L) used (L) volume (L)

Months July 1994 108 174 -66 August 1994 180 102 78 September 1994 54 66 -12 October 1994 180 150 -30 November* 1994 198 186 -12 December 1994 120 120 0 (up to the 21 st) January 1995 155.5 151.5 4 February 1995 118 146 -28 March 1995 228 247 -19 April 1995 185 206 -21 May 1995 130 72 +58 June 16th 1995 0 0 0 * in the month of November, the culture room had an AC problem which contributed to loss of culture.

Ill. ECOLOGICAL INTERACTION STUDIES

A. Brevetoxin Ingestion in Black Seabass

1) Materials and Methods

2 After four days of feeding, the fish were sacrificed. Ten g of muscle tissue and all of the digestive tract were collected and analyzed separately for brevetoxin accumulation, according to the procedure described below. Samples of shrimp tissue spiked with 50 µg of brevetoxin also were analyzed to determine the fate of brevetoxin in shrimp tissue. Duplicate samples of spiked shrimp were extracted immediately after and at 30 minutes after the brevetoxin was added to test toxin recovery.

a) Tissue Extraction Method

Acetone Extraction Method: Homogenize tissue in acetone for 3 minutes with tissue miser Suction filter sample using a glass fiber filter in a Buchner funnel Add filter cake and filter to beaker and homogenize with acetone again Filter again and transfer filtrate to a round bottom-boiling flask Rotary evaporate until only the watery residue remains Mix residue w/ca 5 ml of methylene chloride and transfer to a 60 ml separator-y funnel Rinse flask w/ca 5 ml of HPLC water and transfer to the sep funnel Rinse flask w/ca 5 ml methylene chloride and transfer to the sep funnel (2x) Add ca. 1 ml of saturated NaCl to separator-y funnel, shake and let phases separate Drain methylene chloride through sodium sulfate into a 50 ml pear shaped flask Rinse boiling flask w/ca 5 ml methylene chloride and transfer to sep funnel Shake and let phases separate Drain methylene chloride through sodium sulfate into the 50 ml pear shaped flask Evaporate sample down to 1 ml Prepare first column using 10 cm of 5% deactivated silica topped w/3 cm of sodium sulfate Introduce sample to column and rinse w/30 ml methylene chloride Elute toxins into a 125 ml pear shape w/20 ml of 80:20 ethyl acetate: methylene chloride Rotary evaporate sample till dry, reconstitute in 1 ml methylene chloride Prepare second column sample using 7 cm 5% deactivated alumina topped w/2 cm of sodium sulfate Rinse column w/20 ml of methylene chloride Elute toxins w/20 ml ethyl acetate Rotary evaporate till dry and transfer to a graduated test tube in methanol Analyze w/HPLC

Quantitative and qualitative analysis was performed with high performance liquid chromatography (HPLC), using a Varian model 5000, with a 25 cm x 0.5 cm DB5

reverse phase SiO2-C18 column, isocratic mobile phase of 85:15 MeOH:H2O, flow rate of 1 mI/min and uv detector at 215 nm (Pierce et al., 1992). The HPLC instruments were calibrated with brevetoxin standards to verify qualitative retention times and quantitative detector response. The toxin nomenclature used is BTX representing brevetoxin. The structures follow these reported by Poli et al. (1986). The genus and species, Ptychodiscus brevis, was changed back to Gymnodinium breve, in 1989 (Steidinger 1989).

3 2) Results and Discussion

Results of the brevetoxin analyses in shrimp tissue are given in Table 2. Brevetoxin was not readily recovered from the shrimp samples, resulting in about 10% immediate recovery from muscle, and an average of 5% recovery 30 minutes after addition of the toxin, indicating rapid alteration of brevetoxin in shrimp tissue.

Fish in the control tank were fed shrimp spiked with the carrier solvent (methanol) each day for four days and showed no brevetoxin in tissue or in the digestive tract. No brevetoxin was observed in muscle tissue samples from any of the fish, including those fed the highest concentration of 500 µg/day. The digestive track samples indicated a trace (< 10 ng/g) of brevetoxin in one of three fish fed 100 µg toxin each day. One out of two fish fed 250 µg for all four days exhibited brevetoxin accumulation at 0.13 µg/g in the digestive tract, where as the digestive tract from a fish fed 250 µg for two days followed by 500 µg for two days contained 0.33 µg/g.

Only one of the three fish offered shrimp containing 500 µg brevetoxin would eat the shrimp. This fish contained brevetoxin in the digestive tract in the amount of 0.26 µg/g. None of the fish that ate the brevetoxin-spiked shrimp died.

The amount of toxin ingested by these fish ranged from 100 µg/day (400 µg total for four days) to 500 µg/day (2,000 µg total for the four days), which is equivalent to the amount of toxin produced by G. breve in one liter of culture containing 4 x 106 cells/L daily, to 20 x 106 celIs/L daily.

These results show that ingestion of shrimp spiked with brevetoxin had no apparent toxic effect on the fish, and very little was found to accumulate in the digestive tract over the four day feeding period. That the brevetoxin was not recovered from spiked shrimp tissue indicates that the shrimp may have altered the toxin or bound it in such a way that the toxin was unavailable to the fish upon ingestion.

Further investigations are warranted to assess the effects and accumulation of toxins entering the fish’s digestive system unaltered (toxins in association with an inert substance) and toxins naturally ingested by organisms (clams, oysters or zooplankton) subsequently fed to carnivorous fishes.

B. Evaluation of Food Carriers

The inability to recover brevetoxin spiked into shrimp tissue, in the above ingestion experiment indicates, that brevetoxins added to tissue may have been altered in the food carrier prior to ingestion. To avoid pre-ingestion alteration of the toxins, tests to find a non- destructive food carrier were undertaken.

Several food carriers were tested for acceptance and ingestion by the fish (black seabass) and, for recovery of added brevetoxin. Moistened bread balls had no effect on toxin recovery (99% recovery of toxins added to the bread), however, fish did not accept the bread balls, frequently ignoring or spitting them out. Other carriers tested included kelp balls made from kelp powder, plastic gel-caps, bread balls covered with an artificial fish attractant, chum-n- rub, also were rejected by the fish.

4 Commercial fish pellets (Ziggler, Inc., PA), made from a mixture of fish meal, grain and other additives were readily eaten by the fish. Recovery of brevetoxin from fish meal, however proved too difficult for quantitative analysis of toxin content.

Future studies will focus in encapsulated brevetoxin placed within fish meal or shrimp, to enhance feeding without having the toxin come in contact with the food carrier.

Table 2. Brevetoxin Concentration in Brevetoxin Spiked Shrimp and in Black Seabass Muscle Tissue and Digestive Tract Following Ingestion of the Shrimp.

Spiked Shrimp (50 µg):

1 5.2 1.6 2 5.5 3.6

Seabass: Muscle µg/g Control 100 250 250/500 500 A < < < X X B < < X < X C < < < X <

Digestive Tract µg/g A < < 0.13 x X B < T X 0.33 X C < < < X 0.26

Note: - Control ate 7-8 g food all 4 days - 100 ate 100 µg all 4 days - 250 A and C ate 250 µg all 4 days - 250/500 ate 250 µg for 2 days and 500 µg for 2 days - 500 C ate 230 µg day 1 and 500 µg days 2, 3 & 4 - x = Not analyzed, < = Limit of detection 0.05 µg/g

C. First Long Term (14 Day) Clam Exposure With Depuration (2/6/95)

1) Methods

The 14 day exposure began on 2/6/95 using local Quahog clams (Mercenaria campchiensis) collected off the south end Longboat Key on 1/25/95. Thirteen clams were used for this study, each in a 20L glass aquarium equipped with a magnetic stirrer to maintain water circulation. Each was glued to a vertical plexiglass stand using 3M 5200 Marine Adhesive and acclimated to laboratory tanks for ten days. During the acclimation period the clams were fed daily rations of Tetraselmis chuii. After placing them into individual tanks, the temperature was maintained at 25°C (±2°C).

During the first seven days of exposure, viable red tide cells were used to maintain an exposure level of 2.0 x 106 c/L. The nine test clams were fed only red tide; three seven

5 day exposure test clams (7t), three fourteen day exposure test clams (14t) and three seven day exposure test clams with seven day depuration period (7dt). Four control clams were fed daily rations of Tetraselmis chuii for the duration of the experiment (14c). One additional control tank was set up every other day with G. breve but no clam (GBC). Water in tanks was exchanged every 24 hours and re-inoculated to correct concentrations by dilution of G. breve cultures. Samples were taken for cell counts and enumerated by using strip techniques for Sedgwick-Rafter counting chambers (See Appendix “A”). Twice weekly, original culture and outgoing water was taken for toxin extraction. Temperature, salinity, pH and dissolved oxygen levels were measured periodically throughout the exposure.

After seven days of exposure, the three seven day exposure test clams and two control clams were sacrificed for toxin analysis. At this time, the three seven day exposure test clams with seven day depuration were allowed to depurate while the three remaining exposure test clams and the two control clams continued G. breve and Tetraselmis chuii exposure for a total of 14 days. At the completion of the 14 days, all clams were sacrificed for toxin analysis.

2) Results

Water quality parameters and cell concentrations in the clam exposure aquaria are given in Table 3. Temperatures were maintained in a relatively constant range of between 20°C and 25°C. The pH range was between 7.0 and 8.3. It seems that the pH and the cell concentrations appeared to have a direct relationship when the pH was low, the cell concentrations also were low. In other words, when the clams were eating large quantities of G. breve, the pH decreased. The salinity of the sea water stayed consistently between 33ppt and 35ppt with one outlier. The range of the dissolved oxygen was between 3.0mg/L and 9.7mg/L. In many instances, the dissolved oxygen was demonstrated to have a direct relationship with G. breve concentrations. It was noted that when the clams were siphoning much of the time or consuming large amounts of G. breve, the dissolved oxygen would decrease sharply. Based on personal observation, it seemed that the clams siphoned much of the time in the beginning of the experiment, resulting in many feces and low cell counts, but as the experiment progressed, little siphoning was noted with higher cell counts and fewer feces. The number of deposits of G. breve cells on the bottom of the aquaria also increased during the second seven days.

A graph of G. breve cell counts after 24 hours of clam exposure for each day of exposure is given in Figure 1 for the 7 day clam exposure, and in Figure 2 for the 14 day clam exposure. These results indicate a periodical trend, especially noticed for the 14 day test clams (Figure 2) exhibiting a pattern of crests and troughs throughout the experiment.

Results of brevetoxin BTX analyses in water and clams are given in Table 4. These data show very little accumulation of brevetoxins in exposed clams under these experimental conditions.

From these results, a second experiment was designed to include extracting groups of clams at two crests and a trough as they go through this cycling, to determine whether the clams accumulate toxin in a pattern similar to G. breve cell consumption. Also, a technique to try to decrease the number of G. breve deposits was developed by changing the aquaria with a clean one when deposits formed.

6 Table 3. 14 Day Clam Exposure With Depuration (2/6/95) Water Quality and G. breve Concentrations

7 Table 3. (continued) 14 Day Clam Exposure With Depuration (2/6/95) Water Quality and G. breve Concentrations

8 Table 3. (continued) 14 Day Clam Exposure With Depuration (2/6/95) Water Quality and G. breve Concentrations Table 3. (continued) 14 Day Clam Exposure With Depuration (2/6/95) Water Quality and G. breve Concentrations Table 4. Clam Exposure - Bioaccumulation: 14 Day Exposure With Depuration (2/6/95) Brevetoxin concentrations in exposure water and clams

11 Table 4. (continued) Clam Exposure - Bioaccumulation: 14 Day Exposure With Depuration (2/6/95) Brevetoxin Concentrations in Exposure Water and Clams

12 Figure 1. G. breve Test Tank Cell Counts at 24 Hours for 7 Day Clam Exposure (7t), (2/6/95) Day

Figure 2. G. breve Test Tank Cell Counts at 24 Hours for 14 Day Clam Exposure (14t), (2/6/95) D. Second Long Term (14 Day) Clam Exposure (3/21/95)

1) Methods

The second 14 day exposure began on 3/21/95 using local Quahog clams Mercenaria campchiensis collected off the south end Longboat Key on 3/10/95. Twelve clams were used for this study, each in a 20L glass aquarium equipped with a magnetic stirrer to maintain water circulation. Each was glued to a vertical plexiglass stand using 3M 5200 Marine Adhesive and acclimated to laboratory tanks for ten days. During the acclimation period the clams were fed daily rations of Tetraselmis chuii. After placing them into individual tanks, the temperature was maintained at 25°C (± 2°C).

For the fourteen day exposure, viable red tide ceils were used to maintain an exposure level of 2.0 x 106 c/L. The nine test clams (A-l) were fed only red tide. Three control clams (14c) were fed daily rations of Tetraselmis chuii at 10 x 106 c/L. Three additional control tanks were set up and inoculated with G. breve but no clams (GBC). Water in tanks was exchanged every 24 hours and re-inoculated to correct concentrations by dilution of both cultures. Tanks which developed deposits were removed and replaced with a cleaned tank. Both G. breve and Tetraselmis samples were taken for cell counts and enumerated by using strip and field techniques for Sedgwick-Rafter counting chambers (See Appendix “A”), respectively. Original culture and outgoing water was taken for toxin extraction at random. Temperature, salinity, pH and dissolved oxygen levels were measured periodically throughout the exposure.

After six days of exposure, three test clams were sacrificed for toxin analysis. It appeared that the clams had consumed G. breve because the test tanks showed low cell counts. On day seven, three more test clams were taken after cell counts proved that these clams were no longer eating. At 14 days, the final three clams were sacrificed for toxin analysis because they resumed G. breve consumption. The three Tetraselmis fed control clams were also sacrificed on the last day of the experiment.

b) Results

Water quality parameters and cell concentrations in the clam exposure aquaria are given in Table 5. These results show that the temperatures were maintained in a range between 23°C and 27.5°C. The pH range was between 7.6 and 8.2. The salinity of the sea water stayed consistently between 35ppt and 36ppt. The range of the dissolved oxygen was between 4.8mg/L and 8.8mg/L. On a personal observation, overall it seemed that the clams siphoned less than in the previous experiment. Because of this, it was difficult to determine when to take the clams for toxin extraction. From the graph of data, (Figure 3 and Figure 4), it was observed that the clams did not exhibit any cyclical feeding behavior.

Results of brevetoxin (BTX) analyses in water and clams are given in Table 6. These data show no accumulation of brevetoxins in exposed clams.

15 Table 5. Second 14 Day Clam Exposure (3/21/95) Water Quality and G. breve Cell Concentrations

16 Table 5. (continued) Second 14 Day Clam Exposure (3/21/95) Water Quality and G. breve Cell Concentrations

17 Table 5. (continued) Second 14 Day Clam Exposure (3/21/95) Water Quality and G. breve Cell Concentrations

18 Table 5. (continued) Second 14 Day Clam Exposure (3/21/95) Water Quality and G. breve Cell Concentrations

19 Table 5. (continued) Second 14 Day Clam Exposure (3/21/95) Water Quality and G. breve Cell Concentrations

20 Figure 3. G. breve Cell Counts for Individual Exposure - Tanks After 24 Hours Figure 4. T. chuii Cell Counts for Individual - Control Tank Counts After 24 Hours Table 6. Brevetoxin Concentrations in Exposure Media and in Clams Exposed to Live G. breve Cells (Second Clam Exposure)

< = less than limit of detection = 0.05 µg/L or 0.1 µg/L clam tissue

23 IV. RED TIDE FIELD STUDIES

A. 1994 Red Tide Bloom (9/16/94 - 1/4/95)

A natural red tide bloom was detected in the Gulf of Mexico and in Charlotte Harbor on September 16, 1994. Mote staff initiated monitoring in the Sarasota area on Monday, September 19, 1994 continuing periodic sampling through January 4, 1995.

The distribution and intensity of the red tide bloom in Sarasota Bay and near shore Gulf of Mexico is shown in Table 7, indicating cell counts and sample location. A graph of G. breve cell counts at the New Pass dock is shown in Figure 5. The 1994 red tide bloom developed high concentrations (> 107 c/L), as early as September 20th, in the near-shore Gulf, indicating conditions for immediate fish kill. Concentrations remained high in the Gulf through October 26th, and then diminished to low concentrations (< 105 c/L) by November 4th. Cell counts in Sarasota Bay, however, remained in the low to moderate levels (< 105 and < 106 c/L), indicating that conditions in the Bay were not supportive of an intensive bloom. New Pass samples indicate transport between near-shore Gulf and Bay waters. These concentrations remained in the low-to moderate range (reflecting Sarasota Bay levels) except in December, near the end of the bloom period.

Toxins and pigments associated with natural blooms also were investigated. Samples of water from an intensive patch of the red tide bloom were collected from near surface and from 2 m depth. G. breve cells were enumerated and the samples were analyzed for pigment and toxin content. Results of the cell counts and toxin analyses are given in Table 8. The primary toxins recovered during the natural bloom were BTX-2, -3 and -5. Interestingly, BTX-1 was not present in quantifiable amounts. G. breve collected from near surface water appeared to produce the same amount and type of toxins as those produced by G. breve collected from 2 m depth.

B. Red Tide Bloom (4/13/94 - 6/16/95)

A second red tide bloom was observed in Charlotte Harbor in early April, with first detection of red tide in the Sarasota Area on April 20, 1995.

The distribution and intensity of the bloom for Sarasota Bay and the near-shore Gulf of Mexico are given in Table 9. A graph of G. breve count at New Pass is given in Figure 6.

Red tide cell concentrations for the 1995 bloom exhibited moderate to high concentrations in the near-shore Gulf and in isolated patches in Sarasota Bay early in the bloom development (April 4, 1995). No G. breve celIs were detected at 3 to 7 miles off Sarasota in the Gulf at this time. The cell concentrations remained at moderate levels in the Bay and near-shore Gulf from April 20th through June 7th with periodic, isolated patches of higher concentrations in the Bay and Gulf. Extremely high concentrations were observed in the Bay and Gulf on 6/8, and these repeatedly diminished to none detected by June 12, 1995.

The red tide cell counts at New Pass (Figure 6) reflect the extended period of moderate levels with periodic spikes to high. The extremely high levels in early June are also reflected just prior to a rapid reduction in cell concentrations.

24 Table 7. Red Tide Field Studies (9/19/94 - 1/3/95)

25 26 27 28 29 30 31 32 Table 8. Brevetoxin Analysis from Natural Red Tide Bloom Areas

Pbtx-3 Pbtx-2 Pbtx-5 TOTAL Pbtx-3 Pbtx-2 Pbtx-5 TOTAL G. breve 9/20/94 SAMPLING µg/l µg/l µg/l µg/l µg/MC µg/MC µg/MC µg/MC cell/L x 106

SUR 1 3.3 326 36.9 366.2 0.4 35 3.97 39.37 9.3 SUR 2 3.7 340 42.6 386.3 0.3 30 3.81 34.11 11.2 SUR 3 3.8 310 40.4 354.2 0.4 29 3.77 33.17 10.7 2 METER 1 9.1 490 62.2 561.3 0.7 40 5.01 45.71 12.3 2 METER 2 5.8 430 57.1 492.9 0.5 37 4.92 42.42 11.6 2 METER 3 4.5 188 24.8 217.3 0.6 26 3.49 30.09 7.2 9/29/94 SAMPLING

SUR1A 4.29 113 16 133.29 0.69 18.1 2.57 21.36 6.2 SUR1B 10.5 113 16.7 140.2 1.98 21.1 3.14 26.22 5.3 SUR1C 5.01 109 15.3 129.31 0.83 17.7 2.5 21.03 6.2 SUR2 6.8 102 11.9 120.7 1.37 20.6 2.4 24.37 5.0 SUR3 11.5 94.4 12.6 118.5 1.89 15.4 2.06 19.35 6.1 2 METER 1 7.93 88.2 12.6 108.73 1.3 14.4 2.06 17.76 6.1 2 METER 2 5.88 92.4 15.7 113.98 1.36 21.3 3.62 26.28 4.3 2 METER 3 1.03 89.5 13.5 104.03 0.24 20.7 3.11 24.05 3.5

MC = Million Cells

Table 9. (continued) Red Tide Field Studies (4/13/95 - 6/16/95)

35 Table 9. (continued) Red Tide Field Studies (4/13/95 - 6/16/95)

36 Table 9. (continued) Red Tide Field Studies (4/13/95 - 6/16/95)

37 Table 9. (continued) Red Tide Field Studies (4/13/95 - 6/16/95)

38 Table 9. (continued. Red Tide Field Studies (4/13/95 - 6/16/95)

39 Table 9. (continued) Red Tide Field Studies (4/13/95 - 6/16/95)

* Samples Taken in Surf at Beach S ppt R = Salinity by Refractometer s ppt SCT = Salinity by Conductivity Meter

See Addendum “A” and Appendix “A”

40 41 C. Red Tide Pigment

Water samples, for pigment analysis, were collected at mid-day from the surface layer and 2 meters depth in bloom patches on four days (9/20/94, 9/29/94, 10/26/94 and 12/6/95). G. breve cells from volumes of 50 to 100 ml of these samples were collected on GF/F (Whatman, Inc.) glass fiber filters. These filter samples were stored in liquid nitrogen until analyzed for pigment content. Pigment extraction from the filter samples involved 20 seconds of sonication in 90% acetone followed by overnight storage in a freezer. The pigment extract was clarified by centrifugation and analyzed immediately by HPLC (Wright et al., 1991). Pigments were detected by light absorption in a photodiode-array spectrophotometer (Shimadzu Scientific Instruments, Inc.).

Figures 7, 8 and 9 illustrate some of the data from the pigment analyses. Pigment content is reported on a cell specific basis in these figures. The chlorophyll a and gyroxanthin-diester content of G. breve at the two sampling depths for each sampling date are shown in Figures 7 and 8 respectively. It is apparent that within the precision of our study there is no difference in the content of these pigments between the two depths. This relationship holds for the other detected pigments (data not shown). However, over time the pigment content per cell decreases. This same pattern of decreasing pigment content with time was observed by Millie et al. (1995) in laboratory cultures of G. breve. In their study the pigment shift coincided with transition from log growth phase to stationary growth phase. We hypothesize that actively dividing cells (log growth phase) maintain higher pigment content than cells that are not dividing (stationary growth). This relationship holds potential for use as a way to rapidly assess the growth phase of natural blooms. Figure 9 clearly illustrates that the ratios between the content of one pigment to the content of the other pigments were constant through time. Figure 9 includes only data from 2 m depth, but from the close comparison between the two depths in Figures 7 and 8 this constancy of pigment ratios also holds for the surface samples.

Further analyses are needed on these data. Detailed comparisons with the laboratory data of Millie et al. (1995) may strengthen our hypothesis concerning pigment content and growth phase. The pigment content of laboratory cultures that have been in stationary growth phase for approximately six months is presently being analyzed. Also, quantitative analysis of the relationship between toxin content and pigment content is underway.

D. Bacteriological Studies

1. Heterotrophic Bacteria

Quantitation of “total” heterotrophic bacteria

A total of 67 water samples have been included from both bloom and non-bloom conditions and from both Sarasota Bay and the Gulf of Mexico. Water was collected in sterile plastic containers from a depth of 15 cm. Water was spread-plated directly (0.1 ml) on Difco Marine Agar and also decimally diluted in sterile seawater with 0.1 ml plated. After incubation at room temperature (ca. 22°C) for 3-5 days, colonies were counted. The results are given in Table 10.

42 Plate counts ranged from 110/ml to 3x104/ml. Count distribution was as follows:

Plate count/ml Number of Samples < 103 50 10 3- 104 15 > 104 2

Twenty-five samples were collected from non-bloom conditions with an average plate count of 3,100/ml. Forty-one samples came from waters affected by a G. breve bloom and the average heterotrophic plate count was approximate 600/mI or roughly 1/5 of non-bloom counts. The suggestion is that a red tide may influence total bacterial counts while the bloom is in progress. This could be a result of either a direct inhibition of indigenous marine bacteria or the destruction or alterations of sources of organic matter which normally sustain bacteria (i.e., phytoplankton, zooplankton). These observations require additional field and laboratory confirmation but could represent additional biological/chemical implications of the red tide phenomenon. There are no obvious statistical correlations between plate counts and G. breve counts but this needs further clarification because both counts were not always recovered for the same sample and it is well recognized that both bacterial and dinoflagellate counts can vary significantly in time and in space.

Qualitation of “total heterotrophic” bacteria

A total of 206 bacteria were selected at random from “total” heterotrophic plate count plates (Difco Marine Agar). Isolates were established in pure culture and subjected to the following tests or reactions: Gram reaction, cytochrome oxidase, motility, catalase, oxidative/fermentation response, and sensibility to the vibriostal 0/129 and other appropriate antibiotics. Assignment to genus or group was based on reference to several published identification schemes.

The data (Table 11) show that, qualitatively, there are no observable differences between genera/groups of hetrotrophic bacteria recovered from bloom and non-bloom waters. If red tides have a qualitative effect on hetrotrophic plate count bacteria as suggested above, the observations here indicate that the reduction seems to be “across the board,” i.e., no genera appear to be selectively reduced on Marine Agar counts at room temperature.

2. Vibrio spp.

Quantitation of Vibrio spp.

Two procedures were used to estimate the numbers of potentially pathogenic vibrios:

a) direct plating of water samples of TCBS agar, which is selective for Vibrio spp. and;

b) inoculation of 10, 1.0, 0.1, and 0.01 ml volumes of water into alkaline peptone broth (pH8.5) in a three-tube decimal dilution series (MPN; most probable number) followed by streaking of tubes showing turbidity on TCBS agar.

43 In both procedures, representative isolates on TCBS were further identified to species. All incubations were at 37 C.

A total of 51 water samples were studied; 21 were collected during red tide conditions and 30 were taken under non-bloom conditions. In both cases, (Table 12.) approximately 50% of the samples showed higher Vibrio counts when TCBS agar was inoculated directly with water compared with MPN data. However, water samples taken during non-bloom conditions yielded MPN counts higher than TCBS agar counts in 37% of tests whereas, only 10% of samples collected in blooms showed higher MPN counts. This suggests that, in a red tide, there may be fewer potentially pathogenic vibrios present. Using counts of TCBS agar, 18 samples from non-bloom waters showed an average of approximately 150 vibrio/ml of water while 31 samples taken from red tide waters yielded an average of 56 vibrios/ml; about a third fewer. Although statistical analyses of these data have not been completed, these results suggest that G. breve blooms may reduce the populations of indigenous Vibrio spp., about 12 species of which can caused disease or infections in humans.

This observation confirms those for plate count bacteria which also appear to be less in the dinoflagellate bloom. This may be reflected in lower numbers of the potentially pathogenic vibrios, normally recovered by incubation at 37C on TCBS plate counts or MPN tubes, but not reduced numbers of vibrios recovered at room temperature on Marine Agar (see above).

Qualitation of Vibrio spp.

Colonies varying in size, color and consistency were selected from direct platings on TCBS agar. Positive (turbid) tubes of alkaline peptone broth from the MPN series were streaked for confirmation on TCBS agar and subsequent colonies chosen as above. Isolates were maintained on Marine Agar slants and subjected to the following tests or reactions: Gram reaction, cytochrome oxidase test, color on TCBS medium, reaction in oxidative/fermentative medium, argenine dihydrolase, lysine and ornithine decarboxylase, growth in 0, 8, and 10% NaCl broth, and, where appropriate, indole production, acetone formation, and utilization of mannitol and allobiose. Identification of species was accomplished by reference to several published identification schemes.

A total of six separate species were recovered, plus unidentified. The data indicate that several taxa are recovered in quite different frequencies, from bloom and non-bloom waters using direct plating and broth (MPN) enrichment. Vibrio alginolyticus was found over twice as often with the MPN technique from non-bloom waters but about as frequently with blood agar and both methods in bloom waters. Vibrio damsuela occurred more often in both types of waters using the broth (MPN) enrichment. Conversely, V. vulnificus was found more than twice as frequently from bloom and non- bloom waters by direct plating on TCBS. Clearly, neither method is appropriate for recovery of all species.

44 Date

Figure 7. Chlorophyll a content of G. breve at Two Sampling Depths During a Natural Red Tide Bloom. September - December, 1994

45 Figure 8. Gyroxanthin-diester Content of G. breve at Two Sampling Depths During a Natural Red Tide Bloom. September - May, 1994

46 Figure 9. Comparison of the Concentration of Three Pigments with Chlorophyll-a Concentration in G. breve Collected at 2m Depth During a Natural Red Tide Boom,

47 Table 10. Bacteriological Samples Collected From Gulf and Bay Waters for the 1994/1995 Red Tide Project

“Total” Vibrio Vibrio MPN/ Date Sample site plate count/ml G. breve/L plate count/ml 100 ml

5/19/94 SB - Buttonwood Harbor 4,100 150 > 1,100 5/19/94 SB - MML Dock 4,000 120 > 1,100 5/19/94 Big Pass 11,000 250 > 1,100 7/26/95 SB - MML Dock 30,000 3000 4,600 7/26/94 SB - County Club Shores 3,100 400 4,600 7/26/94 Big Pass 7,000 < 100 > 46,000 7/26/94 New Pass 3,100 300 > 1,100 1/17/95 New Pass 500 10 240 1/20/95 SB - MML Dock 800 20 460 1/25/95 New Pass 430 10 240 1/31/95 New Pass 350 20 240 2/3/95 SB - MML Dock 2,200 < 10 460 2/9/95 New Pass 1,900 10 240 2/14/95 GM - 7 Miles Out 110 <10 23 2/14/95 GM - 5.5 Miles Out 1,000 <10 23 2/14/95 GM - 4.5 Miles Out 1,500 <10 23 2/14/95 GM - 3 Miles Out 860 <10 23 2/23/95 New Pass 860 <10 240 3/2/95 New Pass 910 <10 460 3/10/95 New Pass 510 <10 240 3/17/95 New Pass 350 <10 1,100 3/22/95 SB - MML Dock 120 <10 240 3/30/95 New Pass 830 10 460 4/7/95 New Pass 700 20 1,100 4/20/95 New Pass 740 130 4,600 4/21/95 SB - MML Dock 1,000 0 130 930 4/21/95 New Pass 740 421,000 30 2,400 4/21/95 SB - North of MML 700 540,000 80 4,600 4/21/95 NP - West of MML 640 290,000 10 4,600 4/24/95 New Pass 870 > 1,000,000 30 2,400 4/25/95 SB - MML Dock 1,100 21,000 170 >11,000 4/26/95 New Pass 290 392,000 20 430 4/26/95 GM - 100 Yards Off LBK 400 205,000 <10 4,600 2 Miles North of NP 4/26/95 GM - New Pass 210 200,000 70 4,600 4/26/95 GM - Off NP 380 210,000 <10 430 4/27/95 Siesta Key Shore 410 <10 ND 4/27/95 Siesta Key - 2 Miles Out 330 29,000 20 ND 4/27/95 Siesta Key - 1/2 Mile Offshore 160 392,000 10 ND 4/27/95 Lido Beach 310 119,000 20 ND 4/27/95 Big Pass 850 280,000 50 ND 4/27/95 LBK - Far Horizons Beach 440 6,400,000 <10 ND 4/27/95 Mid LBK - 2 Miles Out 600 744,000 <10 ND 4/27/95 SB - ICW Near Library 620 284,000 40 ND 4/27/95 SB - ICW Marina jacks 560 1,154,000 40 ND 4/29/95 New Pass Contaminated 130 >11,000

* NP- North Port, LBK- Longboat Key, ICW- Intercostal Waterway, SB - Sarasota Bay, GM-Gulf of Mexico, MML-Mote Marine Laboratory

48 “Total” Vibrio Vibrio MPN/ Date Sample site plate count/ml G. breve/L plate count/ml 100 ml

5/1/95 New Pass 630 133,000 40 1,500 5/2/95 New Pass 800 83,000 40 >11,000 5/5/95 SB - MML Dock 1,400 482,000 200 4,600 5/8/95 New Pass 840 72,000 80 11,000 5/8/95 Mid LBK - 1/2 Mile Out 210 194,000 10 ND 5/8/95 Mid LBK - 100 Yards Offshore 240 10 ND 5/8/95 SB - Off Ringling Bridge 520 165,000 40 ND 5/8/95 Lido Beach - 800 Yards Offshore 210 93,000 20 ND 5/8/95 Little SB 1,000 230,000 10 ND 5/8/95 Siesta Key - Point of Rocks 2,000 510,000 90 ND 5/8/95 North End of Siesta Key 190 575,000 <10 ND 500 Yards Offshore 5/11/95 New Pass 830 604,000 70 4,600 5/16/95 Mid LBK - Shore 340 151,000 40 ND 5/16/95 Mid LBK - 1 Mile Out 230 615,000 <10 ND 5/16/95 Lido Beach 340 449,000 <10 ND 5/16/95 SB - Off Ringling Bridge 750 503,000 100 ND 5/23/95 Siesta Key - Point of Rocks 600 428,000 <10 ND 5/23/95 Big Pass 930 417,000 20 ND 5/23/95 GM - 1 Mile Off NP 260 32,000 <10 ND 5/23/95 LBK - Off Holiday inn 380 845,000 70 ND 5/23/95 Mid LBK - 100 Yards Offshore 710 7,030,000 30 ND 5/23/95 Longboat Pass 780 210,000 <10 ND

* NP- North Port, LBK- Longboat Key, ICW- Intercostal Waterway, SB -Sarasota Bay, GM- Gulf of Mexico, MML-Mote Marine Laboratory

49 Table 11. Genera/Groups of Bacteria Recovered From Plate Counts

Non-bloom Bloom Overall Water Water Group No. % No. % No. %

Acinetobacteria 4 2 1 1 3 2

Aeromonas 34 17 12 15 22 18

Alteromonas/ 62 30 19 24 43 34 Pseudomonas

Cytophaga 3 2 2 3 1 1

Enterobacteriacae 4 2 3 4 1 1

Flavobacterium 1 0.5 1 1

Flavobacterium/ 3 2 3 4 Cytophaga

Fleribacter 4 2 1 3 2

Moraxella 7 3 5 2 2

Moraxella/ 2 1 2 Pseudomonas

Pseudomonas 12 6 6 7 6 5

Serratia 1 0.5 1 1

Vibrio 69 34 26 32 43 34

206 81 125

50 Table 12. Vibrio spp. Recovered From Direct Plating (TCBS) and MPN Tubes

Overall Non-bloom Waters Bloom Waters TCBS MPN TCBS MPN TCBS MPN # % # % # % # % # % # %

Vibrio Alginolyticus 14 19 39 36 4 16 34 43 10 20 5 17

V. damusela 16 12 27 25 3 12 15 19 9 18 12 41

V. flevialis 4 5 12 11 4 16 12 15

V. furnissii 2 2 2 3

V. parahaemolyticus 2 3 3 3 1 4 1 1 1 2 2 7

V. vulnificus 30 41 17 16 11 44 13 17 19 39 1 14

Vibrio spp. 12 16 8 7 2 8 2 3 10 20 6 21 2. Occurrence of Bacteria in Laboratory Cultures of G. breve

Studies have continued on several bacteria which occur in f/2 dinoflagellate cultures. In general, three bacteria have been isolated routinely and include an unpigmented colony type which is quite grainy in appearance, a bright orange-pigmented type and a bright yellow-pigmented biotype. Other isolates from deposits in spent (“crashed”) carboy cultures have also been recovered recently and are being examined morphologically and physiologically.

E. Brevetoxin Analysis in Marine Organisms Exposed to Sublethal Levels of the 1994 Natural Red Tide Bloom.

Because of the long-term duration and consistent, low levels of red tide cells in Sarasota Bay, this bloom provided an opportunity to investigate bioaccumulation of brevetoxins in naturally exposed organisms. Specimens of sea squirt Styela plicata and oyster Crassostria virginica were collected periodically from the Mote dock pilings in Sarasota Bay, an area exposed to sublethal concentrations of red tide cells over an extended period of time. Mullet were collected from the same area of Sarasota Bay. Brevetoxin analysis was performed according to the method described above for black seabass.

Results of these studies are given in Table 13, showing brevetoxin accumulation in sea squirts and oysters. Mullet samples from 10/5/94 did not indicate the presence of brevetoxin, however, mullet collected on 11/16/94 did exhibit small concentrations of brevetoxin in the digestive tract by both HPLC and receptor binding assays. Previous studies have shown the mullet tissue rapidly alters the parent brevetoxin so that it is not readily detected by solvent extraction/HPLC analysis. Brevetoxin concentrations in oysters and seasquirts are shown in Figure 10.

Verification of brevetoxin in the above invertebrates was provided by receptor binding assay by T.A. Leifield and F.M. Van Dolah of the NOAA National Marine Fisheries Laboratory Charleston, South Carolina. The brevetoxin receptor binding assay was based on competition between 3[H]BTX-3 and BTX-3 standards or test sample for voltage-dependent sodium channels in rat brain synaptosome. Receptor binding assays are compared to HPLC brevetoxin analyses (Table 13), showing excel lent agreement.

52 Table 13. Oyster, Seasquirt and Mullet Analysis for 9/94 - 1/95 Red Tide Natural Bloom

BTX 2+3 1HPLC 2 Receptor Binding Wt. Extracted µg/sample

9/23/94 oyster 1 12.54 < N/A oyster 2 12.22 < N/A oyster 1 16.5 < N/A seasquirt 1 47.27 < N/A seasquirt 2 44.4 < N/A seasquirt 3 68.7 0.04 N/A

9/27/94 oyster 1 9.31 0.05 N/A oyster 2 11.7 < N/A oyster 3 10.17 < N/A oyster sp 1 10.2 83% N/A oyster sp 2 10.42 50% N/A seasquirt 1 50.0 0.04 N/A seasquirt 2 41.09 < N/A

10/4/94 oyster 1 15.0 0.05 N/A oyster 2 9.15 < N/A oyster 3 15.03 < N/A seasquirt 1 50.83 0.01 N/A seasquirt 2 54.28 < N/A seasquirt 3 61.3 0.08 N/A

10/5/94 mullet muscle 1 31.46 N/A mullet muscle 2 28.43 N/A mullet muscle 3 28.14 N/A mullet visc 1 30.71 N/A mullet visc 2 23.31 N/A mullet visc 3 34.48 N/A

10/11/94 oyster 1 13.0 0.03 N/A oyster 2 10.7 < N/A oyster 3 22.3 0.02 N/A seasquirt 1 75.22 0.02 N/A seasquirt 2 44.56 < N/A seasquirt 3 37.66 < N/A

< = 0.01 53 Table 13. (continued) Oyster, Seasquirt and Mullet Analysis for 9/94 - 1/95 Red Tide Natural Bloom

BTX 2+3 1HPLC 2 Receptor Binding Wt. Extracted µg/sample

seasquirt 4 sp 22.89 96% N/A seasquirt 5 sp 37.5 113% N/A

10/18/94 seasquirt 1 62.3 0.005 0.03 seasquirt 2 80.8 < 0.01 seasquirt 3 34.9 0.04 0.04

10/19/94 oyster 1 20.3 0.03 0.06 oyster 2 18.3 0.05 0.12 oyster 3 20.8 0.07 0.13 oyster 4 sp 26.3 40.6 2.32 oyster 5 sp 21.9 37.0 1.58

10/25/94 seasquirt 1 57.0 0.07 0.04 seasquirt 2 80.2 0.02 0.02 seasquirt 3 102.0 0.03 0.03 seasquirt 4 sp 69.9 4.0 0.23 seasquirt 5 sp 57.9 4.4 0.11

10/27/94

oyster 1 18.54 0.07 0.13 oyster 2 15.34 0.04 0.09 oyster 3 18.99 0.07 0.10 clam 1 29.8 < < clam 2 29.8 < < clam 3 9.03 < <

11/1/94 seasquirt 1 54.37 N/A 0.08 seasquirt 2 81.7 N/A 0.04 seasquirt 3 64.4 N/A 0.03

11/2/94 oyster 1 16.3 0.05 0.12 oyster 2 18.99 0.03 0.08

< = 0.07 54 Table 13. (continued) Oyster, Seasquirt and Mullet Analysis for 9/94 - 1/95 Red Tide Natural Bloom

BTX 2+3 1HPLC 2 Receptor Binding Wt. Extracted µg/sample

oyster 3 14.56 0.08 0.10

11/7/94 clam tiss 1 19.78 < clam tiss 2 12.0 < clam tiss 3 3.65 < clam dt 1 33.1 < clam dt 2 26.79 < clam dt 3 9.6 <

11/8/94 seasquirt 1 61.5 0.06 0.04 seasquirt 2 66.2 0.01 N/A seasquirt 3 60.3 0.03 0.03

11/10/94 oyster 1 16.73 0.04 0.08 oyster 2 21.03 < 0.15 oyster 3 22.44 0.03 0.08

11/14/94 seasquirt IN3 1 6.39 < 0.17 seasquirt IN 2 7.4 < N/A seasquirt IN 3 5.7 24 N/A seasquirt OUT4 1 45.8 0.03 0.02 seasquirt OUT 2 29.1 0.03 N/A seasquirt OUT 3 43.2 21.0 0.78

11/15/94 oyster 1 14.46 0.11 0.38 oyster 2 17.43 0.14 0.14 oyster 3 22.67 0.07 0.10

11/16/94 mullet muscle 1 39.7 < 0.02 mullet dig tract 1 32.93 0.06 0.02 mullet muscle 2 32.3 < N/A

< = 0.07 55 Table 13. (confirmed) Oyster, Seasquirt and Mullet Analysis for 9/94 - 1/95 Red Tide Natural Bloom

BTX 2+3 1HPLC 2 Receptor Binding Wt. Extracted µg/sample

mullet dig tract 2 21.89 .86 0.86 mullet roe 2 21.26 < 0.08

11/21/94 seasquirt IN 1 7.0 0.17 0.20 seasquirt OUT 1 45.8 0.05 0.05 seasquirt IN 2 4.7 0.06 0.14 seasquirt OUT 2 29.1 0.07 0.10 seasquirt IN 3 5.7 0.08 0.13 seasquirt OUT 3 43.2 0.01 0.06

11/22/94 clam tiss 1 11.36 N/A clam tiss 2 14.27 N/A clam tiss 3 2.8 N/A clam dt 1 28.94 0.10 clam dt 2 38.19 0.04 clam dt 3 6.15 N/A

11/23/94 oyster 1 14.03 0.11 0.10 oyster 2 21.66 0.19 0.07 oyster 3 19.28 0.09 0.14

11/29/94 seasquirt IN 1 8.6 0.16 0.18 seasquirt IN 2 10.04 0.10 0.17 seasquirt IN 3 sp 4.18 3.6 12.55 seasquirt IN 4 18.8 0.03 N/A seasquirt OUT 1 62.5 0.06 0.02 seasquirt OUT 2 48.5 0.04 N/A seasquirt OUT 3 sp 35.3 1.6 0.17 seasquirt OUT 4 75.3 0.01 N/A

11/30/94 oyster 1 13.72 0.07 0.11 oyster 2 19.69 0.07 0.08

< = 0.07 56 Table 13. (continued. Oyster, Seasquirt and Mullet Analysis for 9/94 - 1/95 Red Tide Natural Bloom

BTX 2+3 1HPLC 2 Receptor Binding Wt. Extracted µg/sample

oyster 3 25.86 0.04 0.10

12/5/94 oyster 1 12.8 oyster 2 16.39 oyster 3 sp 19.09 oyster 4 13.02 12/6/94 seasquirt IN 1 7.17 0.11 0.20 seasquirt IN 2 9.39 0.08 0.29 seasquirt IN 3 10.38 0.08 0.22 seasquirt OUT 1 73.1 < N/A seasquirt OUT 2 69.1 0.01 0.02 seasquirt OUT 3 79.4 < 0.01

12/8/94 clam tiss 1 7.8 N/A clam tiss 2 10.93 N/A clam tiss 3 12.28 N/A clam dt 1 30.16 N/A clam dt 2 26.11 0.06 clam dt 3 36.54 0.05

12/9/94 oyster 1 N/A 0.13 0.14 oyster 2 N/A 6.19 0.09

12/13/94 seasquirt IN 1 11.67 0.28 0.33 seasquirt IN 2 6.25 < N/A seasquirt IN 3 6.03 0.20 0.27 seasquirt OUT 1 83.68 0.01 N/A seasquirt OUT 2 67.81 0.01 0.13 seasquirt OUT 3 65.03 0.01 0.06

12/14/94 oyster 1 12.79 0.19 N/A oyster 2 17.82 0.06 0.15 oyster 3 18.62 0.03 0.15

< = 0.01 57 Table 13. (continued) Oyster, Seasquirt and Mullet Analysis for 9/94 - 1/95 Red Tide Natural Bloom

BTX 2+3 1HPLC 2 Receptor Binding Wt. Extracted µg/sample

12/15/94 mullet tiss 1 17.06 0.02 N/A mullet tiss 2 18.16 < N/A mullet tiss 3 14.8 < N/A mullet liver 1 1.95 0.18 N/A mullet liver 2 4.34 0.04 N/A mullet liver 3 3.72 < N/A 12/19/94 seasquirt IN 1 0.30 N/A seasquirt IN 2 5.58 < 0.10 seasquirt IN 3 sp 4.08 16.0 1.92 seasquirt IN 4 6.21 0.16 0.17 seasquirt OUT 1 45.8 0.03 0.02 seasquirt OUT 2 55.31 0.03 0.02 seasquirt OUT 3 sp 67.97 5.7 N/A seasquirt OUT 4 84.58 0.02 0.02

12/20/94 oyster 1 16.25 0.12 0.14 oyster 2 20.67 0.17 0.06 oyster 3 sp 15.06 24 N/A oyster 4 16.38 0.04 0.15

12/22/94 mullet tiss 2 N/A < N/A mullet tiss 3 N/A < N/A mullet liv 2 N/A 0.04 N/A mullet liv 3 N/A < N/A

1/3/95 seasquirt IN 1 6.57 0.19 0.10 seasquirt IN 2 11.19 0.04 0.08 seasquirt IN 3 7.69 0.15 N/A seasquirt OUT 1 74.47 0.09 0.03 seasquirt OUT 2 35.79 0.13 0.04 seasquirt OUT 3 37.3 0.05 N/A

1/4/95 oyster 1 14.09 < 0.16

< = 0.07 58 Table 13. (continued) Oyster, Seasquirt and Mullet Analysis for 9/94 - 1/95 Red Tide Natural Bloom

BTX 2+3 1HPLC 2 Receptor Binding Wt. Extracted µg/sample

oyster 2 15.15 < 0.11 oyster 3 17.38 0.03 0.12

1/9/95 seasquirt IN 1 9.15 < N/A seasquirt IN 2 4.44 < N/A seasquirt IN 3 4.8 < N/A seasquirt OUT 1 59.76 0.08 N/A seasquirt OUT 2 51.7 < N/A seasquirt OUT 3 40.63 0.01 N/A 1/10/95 oyster 1 17.81 < N/A oyster 2 18.98 < N/A oyster 3 17.56 < N/A

1/16/95 seasquirt IN 1 4.75 0.06 N/A seasquirt IN 2 9.43 < N/A seasquirt IN 3 4.86 < N/A seasquirt OUT 1 74.47 < N/A seasquirt OUT 2 35.79 < N/A seasquirt OUT 3 37.3 0.01 N/A

1/17/95 oyster 1 16.59 < N/A oyster 2 20.57 < N/A oyster 3 15.34 < N/A

1. HPLC - Analysis at MML 2. Receptor binding assay at NMFS 3. IN = Inside (viscera) of seasquirt 4. OUT = Outside portion of seasquirt

< = 0.01 59 Figure 10. Red Tide Toxin (BTX) in Oysters and Seasquirts During a Natural Bloom in Sarasota Bay VI. REFERENCES

1. Pierce, R.H., M.S. Henry, L.S. Proffitt and A.J. deRosset. 1992. Evaluation of solid sorbents for the recovery of polyether toxins (brevetoxins) in seawater. Bull. Env. Contam. Toxicol. 49:479-484.

2. Poli, M.A., T.J. Mende and D.G. Baden. 1986. Brevetoxins, unique activators of voltage- sensitive sodium channels, bind to specific sites in rat brain synaptosomes. Molec. Pharmac. 30: 129-135.

3. Steidinger, K.A. 1989. Species of the Tamarensis/Catenella group of Gonyaulax. In: Proceedings of the 4th international Conference on Toxic Marine Phytoplankton (E. Graneli, B. Sundstrom, L. Edler, D.M. Anderson, Eds.), Elsevier Press, London, p. 11-16.

4. Guillard, R.R.L. and J.H. Ryther. 1962. Studies of Marine plankton diatoms. I. Cyclotella nana (Heustedt) and Petonula conferracea (cleve) grass. Can. J. Microbial. 8: 229-239.

61 APPENDIX “A”

Methods for Obtaining Phytoplankton Cell Counts

Phytoplankton cell counts were conducted with the Sedgwick-Rafter and Fuchs-Rosenthal counting slides, detailed from The Handbook of Phycological Methods by Janet Stein, 1973. A 10x ocular was used for all counts. No dilutions or replicate 1 ml samples were taken. Instead of dilution, counting methods were changed from Sedgwick-Rafter to the hemacytometer for improved accuracy at higher concentration levels. Samples of approximately twenty miIIiliters were preserved using Utermohls’ solution.

Samples counted with 150 or less cells were enumerated by Sedgwick-Rafter Strip Counting method (see Stein, p 300-301). The lower limit of detection is 3600-5400 cells per liter (21.57 x 10ˆ3/6 strips). The following calculation for cells per liter was used:

21.57 = the number of Whipple squares found in the width of a Sedgwick-Rafter counting slide.

x = the mean of cells in six strips counted.

c/L = ( 21.57 x 1000 ml/L) x X

When counting Tetraselmis chuii, the Sedgwick-Rafter Whipple field counting method was used (see Stein, p 301). The lower limit of detection is 32,000 cells per liter ( 1.16 x 10ˆ6/36 fields). The following calculation for cells per liter was used:

= the mean of 36 random fields

0.05 mm2 = the area of the Whipple square

1.16 x 10ˆ6 = (21.57 x 0.05 mm2)2 x 1000 mm2/ml x 1000 ml/L

c/L = x 1.16 x 10ˆ6

Samples containing over 150 cells per strip were enumerated by a hemacytometer with the Fuchs- Rosenthal cell count method. The lower limit of detection is 52,000 c/L (5 x 10ˆ6 / (16 squares x 6 slides)). The following calculations for cells per liter was used:

= the mean count of 6 slides

c\L = ( 16) x 5 x 10ˆ6

The procedure for the hemacytometer with Fuchs-Rosenthal ruling cell count method is as follows:

A. Hemacytometer Cleaning and Preparation:

1. Remove hemacytometer and cover slip from the storage container.

2. Rinse first with tap water and then with double distilled water.

62 3. Blot dry with a paper towel.

4. Visually make sure that the chambers and entry slots are completely dry by gently wiping with a kimwipe.

5. Wipe bottom of slide under chambers clean of moisture or debris.

6. Wipe cover slip with a kimwipe to remove any remaining moisture or debris.

7. Place cover slip on top of hemacytometer so that it is resting on both pillars and is covering the entry slots equally on both chambers.

B. Sample Preparation:

1. Obtain representative sample from culture container after swirling and gently agitating culture.

2. Place approximately 20 ml of the sample in a vial.

3. Add one small drop of Utermohl’s fixative solution to the sample via a Pasteur type pipet.

4. Cap the sample vial and invert approximately six times to mix the sample well.

C. Placing Aliquot in Chamber:

1. Immediately after mixing the sample vial, withdraw an aliquot into the Pasteur pipet.

2. Discard the first three drops of the aliquot in the pipet.

3. Place the tip of the pipet into the entry slot of the first chamber.

4. Holding the pipet at roughly a 30 degree angle, release a small amount of the sample into the chamber slot until the liquid covers the entire chamber.

5. Discard remaining sample in the pipet.

6. Repeat steps B-4 through C-5 for remaining chambers.

D. Cell Counting:

1. Place the hemacytometer on the compound microscope stage and view under the 10x power objective.

2. Count all sixteen large 1 mm squares. Adopt a consistent method of counting. Follow a zig-zag pattern by starting at the top left square and count over to the right then down and over to the left. Continue until the last square on the bottom row left square is reached. Only count cells that are on the outside border lines for the top row and right hand side edge. Disregard any cells that are on the outside border lines for the left hand side and bottom edges. Record number counted.

3. Repeat step D-2 for all remaining chambers.

63 E. Conversion to Cells/L:

1. Take the total number of cells counted for all chambers and divide by the number of counts made. this will give the average number of cells counted for each chamber.

2. Divide this number by 16 to obtain the average number of cells per 1 mm square.

3. Multiply by 5x10ˆ6 to obtain average number of cells/L.

F. Clean-Up:

1. Rinse all hemacytometer chambers and cover slips with double distilled water and wipe clean according to the instructions in steps A-3 through A-7 if further counts are to be made.

2. If no further counts are made, just rinse with double distilled water and return to appropriate storage containers.